The overall objective of the proposed research is the development, implementation and testing of novel methods for the analysis of human and other complex eukaryotic genomes. There are three specific aims: 1) Development of Streptomyces as a host for the cloning and study of large segments of chromosomal DNA, and concomitant development of methods that utilize special features of a Streptomyces site- specific recombination system for the ordering and analysis of particular genetic regions within the cloned DNA. 2) Development of methods for identifying genomic DNA sequences included within particular replication domains of mammalian chromosomes, elucidation of the chromosomal, organization of replication domains, and analysis of the relationship of replication domains to the genetic map. 3) Development of methods for defining functional transcriptional units within mammalian genomes utilizing a beta-galactosidase-based reporter gene system functionally analogous to the Mu-dlac system employed for the analysis of gene expression in bacteria. Collectively, the proposed investigations represent a multi-faceted major effort of my laboratory to develop and test methodology and research tools that will facilitate elucidation of the structural and functional organization of complex genomes.